11 replies to this topic

[Rose2004]

HI, all,

I saw lysis buffer recipe which contains 100uM Na3VO4 and 25mM beta-glycerophosphate, someone can tell me what Na3VO4, beta-glycerophosphate are for respectively?

I am new in the protein field, Thanks.

[marc_U7snurp]

Hiya!
Some factors that influence the efficiency of solubilisytion and subsequeunt immunoprecipitation of proteins are ionic strength and pH of the Lysis buffer, and the concentration of detergent used.

I use as well 100 uM salt, but KCl.

For the glyc.-phosphate I have no idea!

What kind of cells do you want to lyse?


Cheerz,
Marc

[Rose2004]

The paper which I read is to lyse yeast cells, and the downstream applycation is to do protein-protein binding, add the extract protein (G protein beta subunit in the lysis buffer below)to GST-G alpha subunit bound RESIN, and binding for 10h. The lysis buffer is,

50mM Hepes(PH=7.6)
150mM NaCl
10mM MgCl2,
1mM EDTA
1mM EGTA
25mM BETA-glycerophoaphate
100uM Na3VO4
protease inhibitor cocktail

Thanks for your response,

[bob1]

Hi

Na3Vo4 (sodium orthovanadate) is a phosphatase inhibitor, as is beta-glycerophosphate it appears.

Bob

[Rose2004]

Thanks,Bob,

And another basic question, Do they(sodium orthovanadate and B-glycerophosphate) need to be autoclaved? or use filter?

I used to autocalve everything in order to minium the protease during the protein extract and purification, Is it necessary?

Does detergent(such as NP40, TRIXON 100) need to be autoclaved?filter?

I havent autoclave or filter the PIC.

Thank you, I really appreciate your help.

Edited by Rose2004, 03 March 2005 - 08:21 AM.

[Simonsays]

Sodium orthovanadate is a phosphotase inhibitor and B-glycerophosphate is a false substrate for phosphatase. It keeps phosphatases busy!!

Simon

[bob1]

Hi

I don't know about the autoclaving of Na2VO4 or Beta-glycerophosphate, I would filter to be on the safe side.  However if you have made them up in a clean enough fashion there should be no need to do even this.  Check the product information sheets, these will give you all that sort of info.  

Detergents usually don't need to be filtered or autoclaved, so long as you use sterile techniques to get them out of the bottle.  Another suggestion is to aliquot them and discard if you think you have contaminated them.

Bob

[sharath]

To decide if autoclaving is suitable for a given compound, you need to check its thermal stability. The label generally contains the melting point og the compound. Its also available in the merck index and also in the sigma catalogue.

Autoclaving is done at around 121C. The compound should have a melting temperature atleaset 50C above this point.

[badcell]

Hi. I've never autoclaved NaVO3 nor b-glycerophophate. On the other hand, I found it's important to correctly activate NaVO3 (NaVO3 activation) for maximal activity. I usually make a big batch of it and store it @ -20ºC for years.
Cheers!

[Rose2004]

Thanks a lot for your kindly help!

And more simple questions,

1 Does it always neccesory to add NaVO4 and beta-glycerophosphate to the yeast cell lysis buffer? I read some prototols, all of them have the two stuff.

2 How to activate NaVO4?

Thanks again!

[bob1]

Check out the link in Badcell's reply "(NaVO3 activation)" it works!  This process takes quite a while from memory so keep at it!

Bob

[Rose2004]

Thanks, Bob and Badcell. Got the activation of NaVO4.